The goal of the proposed research is to test the ability of an uncharacterized ubiquitin-like protein family named MUB, for Membrane-anchored Ubiquitin-fold, to funnel activated ubiquitin into critical regulatory reactions at the plasma membrane. How ubiquitin chain length and positioning is determined for poly-, mono-, and multi- ubiquitination at the plasma membrane is an important question in the ubiquitination field, which we will address in the proposed research. Specifically, we plan to define plasma membrane localized interactions between MUBs and the Ubiquitin/26S Proteasome System, ultimately, to better understand the regulation of eukaryotic signal transduction. Many key signaling proteins are regulated by covalent attachment to ubiquitin, a 76 amino acid protein. In many cases, ubiquitination signals protein degradation by the Ubiquitin/26S Proteasome System. The core of the Ubiquitin/26S Proteasome System is extensively characterized and includes a three enzyme cascade - E1, E2, E3, and the large multiproteolytic 26S proteasome itself. It is less well known how the Ubiquitin/26S Proteasome System is coordinated to allow the temporal and spatial resolution incumbent on a system responsible for protein degradation, and other processes including endocytosis, autophagy, peroxisome biogenesis, and DNA repair. MUBs are structurally similar to ubiquitin, but distinguished by a carboxyl terminal signal sequence, called a CaaX box, which recruits a hydrophobic membrane-anchor via the protein prenylation system. Highly conserved MUBs from several model organisms and humans are prenylated in vitro. We propose to test the hypothesis that MUB proteins help to coordinate the activity of the UPS at the plasma membrane. In particular r, protein interaction studies including pull-downs and NMR to characterize interactions in vitro, and fluorescence complementation and co-immunoprecipitation assays to confirm these interactions in vivo will be used. Enzyme activity assays of various Ubiquitin/26S Proteasome System proteins will be performed to determine how they are regulated by MUB proteins. Finally, co-immunoprecipitation assays, mass spectrometry, and genetic analysis will be performed to characterize pathways related to MUB mutant phenotypes. Execution of these aims will reveal new organization and specificity within the Ubiquitin/26S Proteasome System. This research addresses a key area of research because many signaling proteins in the plasma membrane are regulated by ubiquitination, but the mechanisms supporting this process are unknown. The described studies will be conducted in the plant Arabidopsis thaliana for technical reasons. Yet, we expect that our results will be broadly applicable to the highly conserved MUB protein family found across multi- cellular eukaryotes examined to date. PUBLIC HEALTH RELEVANCE: The results of this study will be broadly applicable to the highly conserved MUB protein family found across multi-cellular eukaryotes examined to date. These studies will ultimately contribute to the long term goal of characterizing the cellular organization of the protein degradation pathways, which impact many human health issues including well known diseases such as Huntington's, Parkinson's, Alzheimer's and various cancers.